Advances and Challenges of Using Ovarian Preantral Follicles to Develop Biobanks of Wild Mammals

Abstract

The extinction rate of mammalian species has been accelerated in recent decades. It therefore is important to preserve and store genetic materials in cryobanks for research purposes and subsequent production of offspring through assisted reproductive techniques. Along with the systematic collection and storage of germplasm, research efforts focusing on in vitro culture to produce mature gametes are critical. Specifically, obtaining mature oocytes from in vitro culture of preantral follicles is a great option to enhance female fertility preservation since these early follicles represent 90%–95% of the follicular population on ovarian cortex biopsy. This review presents current advances and discusses limitations and prospects about isolation, cryopreservation/banking, and in vitro culture of preantral follicles from wild species.

Introduction

Conservation of endangered animals is necessary to balance and maintain functioning ecosystems. To mitigate the issues, in situ and ex situ conservation programs are essential, but their success is tightly related to the development of reproductive biotechnologies, especially those geared toward female fertility preservation.¹ Specifically, in vitro culture of oocytes recovered from preantral follicles could serve as a model to understand initial folliculogenesis and could enhance the management of endangered species populations.¹ Knowing that the ovary contains thousands of follicles remaining viable even several hours after the animal's death, collecting and preserving these follicles represent a huge opportunity for germplasm biobanking.²

Preantral follicle development has been extensively studied over the last two decades.² Follicles also can be rescued from ovaries, cryopreserved, and stored indefinitely. When needed, these follicles can be transplanted³ or cultured in vitro⁴ to obtain mature oocytes.¹ Despite some promising results, ideal conditions for cryopreservation and for complete in vitro development of preantral follicles in wild animals still remain a great challenge. The main limiting factors are lack of knowledge on basic reproductive biology of wild species, absence of standardization for protocols used for obtaining follicles, preservation, and culture, as well as species-specific differences.² The objective of this review is to present current advances and discuss limitations and prospects about isolation, cryopreservation/banking, and in vitro culture of preantral follicles from wild species.

Ovarian Follicle Isolation

Preantral follicles can be banked while enclosed into whole ovaries (e.g. in small rodents), in ovarian fragments, or even individually after isolation from the ovarian cortex. In the latter case, mechanical and/or enzymatic methods are used to dissociate follicles from other components of ovarian tissue.⁵

There are three methods for follicle isolation: mechanical, enzymatic, or the combination of both (Table 1). For mechanical isolation, some devices such as a tissue chopper, mixer, surgical scissors, small forceps, or dissecting needles can be used. This method maintains the integrity of follicular structure, basal membrane, and interactions between oocytes/granulosa and theca cells. However, it is relatively slow, laborious, and, in general, provides a low number of follicles.⁶

Table 1.

Application of Different Methods for Ovarian Follicular Isolation in Wild Species

Species	Isolation method	No. of follicles per ovary	Viability (%)	In vitro culture	Viable follicles after cultivation (%)	Authors and year
Cheetah (Acinonyx jubatus)	Mechanical methods using a cell dissociation sieve	980	32.5	—	—	Jewgenow and Stolte (1996)⁷
Jaguar (Panthera onca)	Mechanical methods using a cell dissociation sieve	800	18.7	—	—
Puma (Puma concolor)	Mechanical methods using a cell dissociation sieve	12,500	49.6	14 Days	62.5%
Bengal tiger (Panthera tigris tigres)	Mechanical methods using a cell dissociation sieve	100	26.3	—	—
Siberian tiger (Panthera tigris altaica)	Mechanical methods using a cell dissociation sieve	1,080	18.7	—	—
Sumatran tiger (Panthera tigris sumatrae)	Cell dissociation sieve	1,440	28.6	—	—
Indian lion (Panthera leo persica)	Cell dissociation sieve	600	37.7	—	—
Capuchin monkey (Cebus appeals)	Tissue chopper	−1000 μm: 168,330 ± 17,590; −500 μm: 300,830 ± 111,460; −750 μm: 95,800 ± 31,660; −250 μm: 197,500 ± 50,278	—	—	—	Domingues et al. (2003)¹¹
Gray opossum (Monodelphis domestica)	—	—	—	—	—	Butcher and Ullmann (1996)⁸
Dunnart (Sminthopsis macroura)	—	—	—	—	—	Nation and Selwood (2006)¹⁰
Tammar wallaby (Macropus eugenii)	27-Gauge needles	—	—	4 Days	16%–22%	Richings et al. (2006)⁹
Dasyuridae (fat-tailed dunnart (Sminthopsis crassicaudata) eastern quoll (Dasyurus viverrinus) northern quoll (Dasyurus hallucatus) Tasmanian devil (Sarcophilus harrisii))	0.1% Collagenase and 0.02% DNase, incubated at 35°C for 30 minutes	—	—	Up to 2 days	—	Czarny et al. (2009)¹⁷
Rhesus macaques (Macaca mulatta)	1% de HSA, 0.08 mg/mL de Liberase Blendzyme 3, and 0.2 mg/mL de DNase for 1 hour	—	—	—	—	Xu et al. (2011)¹⁵
Rhesus macaques (M. mulatta)	0.08 mg/mL Liberase Blendzyme 3 with 0.2 mg/mL DNase for 30 minutes	—	—	—	—	Hornick et al. (2012)¹⁶

For wildlife, follicular isolation by mechanical methods using a cell dissociation sieve was initially described for wild felines with varying age. In a specific study,⁷ authors obtained an average of 980 follicles per ovary from cheetahs (Acinonyx jubatus), 800 follicles from jaguars (Panthera onca), 100 follicles from Bengal tigers (Panthera tigris tigres), 1080 follicles from Siberian tigers (Panthera tigris altaica), 1440 from Sumatran tigers (Panthera tigris sumatrae), 600 follicles from Indian lions (Panthera leo persica), and 12,500 follicles from pumas (Puma concolor). Puma isolated follicles were cultured in vitro for 14 days and a mean of 62.5% viable follicles was obtained. Moreover, the authors demonstrated the possibility of recovering considerable amounts of viable preantral follicles from old females, including those with ovarian pathologies (polycystic ovaries).⁷ Another important point is that the authors studied the follicular population of seven wild felines all at once, which is a great advance in knowledge of reproductive physiology of these species.

Another example is in Australian marsupials. Mechanical methods provided isolation of large preantral follicles (120–230 μm) from the gray opossum (Monodelphis domestica).⁸ Large secondary follicles from the Tammar wallaby (Macropus eugenii)⁹ and Dunnart (Sminthopsis macroura)¹⁰ were also obtained, but information regarding follicle size and number was not reported.

Due to the importance of nonhuman primates for biomedical research, techniques for follicle manipulation are better established for them than in any other wild species. A mechanical procedure for isolating preantral follicles using tissue choppers allowed the recovery of 68,330 ± 17,590 follicles from 1/4 ovary in the capuchin monkey (Cebus apella).¹¹ In the rhesus monkey (Macaca mulatta), approximately 50–200 healthy secondary follicles (125–225 μm in diameter) can be mechanically isolated from the ovarian pair.¹² Primate ovarian tissues, however, present dense connective tissue, which renders isolation of individual follicles somewhat difficult without enzymatic treatments.⁵ As a disadvantage, many follicles isolated by this procedure can present basement membrane or theca cell damage, especially when the incubation time is not strictly controlled.¹³ Studies on the rhesus macaque¹⁴ and baboon¹⁵ indicate that preantral follicles can survive and grow after collagenase treatment. Due to variation among animals, it was difficult to uniformly control the level of stromal digestion that has many times led to secondary follicle damage during isolation.¹³ Posteriorly, efficiency of enzymatic methods using association of other enzymes (1% HSA, 0.08 mg/mL Liberase Blendzyme 3 and 0.2 mg/mL DNase for 1 hour; 0.08 mg/mL Liberase Blendzyme 3 with 0.2 mg/mL DNase for 30 minutes)¹⁶ was described to obtain preantral follicles from rhesus macaques. In the latter case, authors defined that association of enzymes can be efficient to isolate follicles without compromising their integrity, allowing availability of more than 500 follicles, ranging in size from 25.0 to 69.6 μm in diameter.¹⁶

Regarding other wild animals, preantral follicles were recovered by enzymatic isolation from ovaries of the fat-tailed dunnart (Sminthopsis crassicaudata), eastern quoll (Dasyurus viverrinus), northern quoll (Dasyurus hallucatus), and Tasmanian devil (Sarcophilus harrisii) using 0.1% collagenase and 0.02% DNase, incubated at 35°C for 30 minutes. Follicles obtained were capable of producing immature oocytes that survived after the vitrification process, presenting a mean of 69.4% ± 2.4% viable oocytes. Given these results, the authors developed a follicle isolation protocol that will play a fundamental role in biobank formation aiming at conserving Dasyurid marsupials.¹⁷

Given the above, comparison of results between species should be analyzed with caution, considering important aspects such as differences in methodology, age of animals used, and species-specific peculiarities. Table 1 summarizes the types of isolation methods and number of follicles obtained from different species of wild animals. Still, a challenge faced by authors is isolation of primordial follicles. This follicular category is important for fertility preservation purposes because although these follicles are most abundant and are present in females of all ages, they are difficult to study due to their small size and susceptibility to dissociation upon isolation.¹⁶ Thus, developing an efficient mechanism to isolate large numbers of primordial and primary follicles or resting follicles is a promising breakthrough that will contribute to utilization of the reproductive potential of rare species.

Cryopreservation

Many studies have been conducted for cryopreservation of preantral follicles from different wild animals with the objective of establishing efficient protocols that allow the maintenance of female gamete viability and maintenance of germplasm banks.¹⁸ Cryopreservation consists of preserving biological material at low temperatures, in most cases cryobiological temperature of liquid nitrogen (−196°C), allowing the cell or tissue to remain viable for indefinite time and to be recovered viable after the thawing process.²

Because of its numerous advantages, cryopreservation using ovarian tissue ensures preservation of female germ cells: first, by ovaries containing many oocytes included in preantral follicles, and second, because these follicles are more resistant to cryoinjury. This resistance is due to the small size of the oocyte; low metabolic rate; stage of cell cycle stopped in prophase I; small amounts of lipids, cortical granules, and support cells; and absence or small development of the zona pellucida.¹⁹

For wildlife, however, cryopreservation of ovarian tissue is still a challenge and protocols have not been optimized, especially due to biological diversity between cell types that add complexity to tissue.¹⁸ To succeed in this biotechnology, some key factors for cell survival must be considered, such as type and concentration of cryoprotectants and the ideal timing for their addition; cooling rate during the freezing process; choice of cryopreservation method (slow freezing or vitrification); and techniques used to ensure removal of cryoprotectant.²

Cryopreservation is performed by two main methods, slow freezing and vitrification (Table 2). Slow freezing, the conventional method, is characterized by exposing cells or tissues to low concentrations of cryoprotectants (∼1.5 M)² for a period ranging from 20²⁰ to 60 minutes.²¹ This method was successfully applied for cryopreservation of agouti (Dasyprocta leporine) ovarian tissue,²² providing 64% of morphologically normal follicles after rewarming. Moreover, in deer (Cervus elaphus hispanicus),²³ kangaroos (Macropus giganteus),⁹ and monkeys,²⁴ slow freezing of ovarian tissue was also able to maintain preantral follicle viability. In wild felids from Asia, slow freezing using ethylene glycol, followed by in vitro culture, allowed obtaining high rates of viable preantral follicles from the Amur leopard (Panthera pardus orientalis), black-footed cat (Felis nigripes), oncilla (Leopardus tigrinus), Geoffroy's cat (Leopardus geoffroyi), northern Chinese leopard (Panthera pardus japonensis), rusty-spotted cat (Prionailurus rubiginosus), serval (Leptailurus serval), and Sumatran tiger (P. tigris sumatrae).⁴ These results confirm that freezing ovarian tissue to form germplasm banks of these species is viable.

Table 2.

Cryopreservation of Ovarian Follicles Derived from Wild Species Using Different Methods

Species	Biotechniques	Cryoprotectants	Results	Authors and year
African elephants and (Loxodonta africana)	Xenograft and cryopreservation of ovarian tissue	1.4 M of DMSO	Presence of primordial and antral follicles after xenograft	Gunasena et al. (1998)⁵⁰
Collared peccaries (Pecari tajacu)	Vitrification of ovarian tissue	3 M of DMSO, EG, and dimethylformamide (DMF)	72% of morphologically normal follicles	Lima et al. (2012)²⁶
Red-rumped agoutis (Dasyprocta leporina)	Slow freezing of ovarian tissue	1.5 M of DMSO, EG, and PROH	DMSO: 60.6 ± 3.6; EG: 64 ± 11.9; PROH: 62 ± 6.9	Wanderley et al. (2012)²²
African lions (Panthera leo)	Cryopreservation of ovarian tissue and in vitro culture	1.5 M of EG	Primordial: 24.5%; primary: 8.2%; secondary: 0.6%	Wiedemann et al. (2013)⁵³
Nonhuman primates	Vitrification and in vitro culture of isolated secondary follicles	Glycerol, EG, DMSO, and synthetic polymers	52% Morphologically normal follicles after thawing and culture	Ting et al. (2013)²⁸
Baboon (Papio anubis)	Vitrification of ovarian tissue and autotransplantation	10% of DMSO and 26% of EG	Primordial: 36%; primary: 68%; secondary: 24%; and antral: 9%	Amorim et al. (2013)²⁹
Spix's yellow-toothed cavy (Galea spixii)	Vitrification of ovarian tissue	3 M of DMSO	70% Morphologically normal follicles	Praxedes et al. (2015)²⁷
Red-rumped agouti (D. leporina)	Xenograft of vitrified ovarian tissue	3 M of DMSO and EG	Fresh control: 61.1% primordial; 14.4% transition; and 15.6% primary; vitrified control: 41.7% primordial and 26.7% primary; xeno-fresh: 63.6% primordial; 9.1% transition; and 9.1% primary; xeno-vitrified: 40% primordial; 7.5% transition; and 12.5% primary	Praxedes et al. (2018)³

DMSO, dimethyl sulfoxide.

The vitrification technique consists of ultrafast reduction of temperature with an average rate of 20,000°C/min–40,000°C/min, using high concentrations of cryoprotectants. This allows reaching an amorphous noncrystallized solid known as the vitreous state, in which parts of molecular chains are disorganized, promoting some molecular mobility.² Among its main advantages are the limited formation of ice crystals due to rapid passage through critical states of temperature reduction (−15°C to 5°C) and rapid processing time. In general, it can be performed in any laboratory or even in the operating room, simultaneously during patient/animal surgery, even in the field, and even immediately after the death of the animal.²⁵

Regarding wild animals, it was recently demonstrated that it is possible to preserve more than 70% morphologically normal preantral follicles after ovarian tissue removal using a solid surface vitrification method in agoutis,³ peccaries (Pecari tajacu),²⁶ and Spix's yellow-toothed cavies (Galea spixii).²⁷ In nonhuman primates, association of ovarian tissue vitrification using combinations of glycerol, ethylene glycol, dimethyl sulfoxide, and synthetic polymers (polyvinylpyrrolidone—PVP) with in vitro culture of isolated secondary follicles preserved preantral follicle morphology (52% ± 2%) and function.²⁸ Moreover, vitrification of Papio anubis ovarian tissue, followed by autotransplantation, resulted in high rates of survival (36% ± 46%—primordial; 68% ± 86%—primary; 24% ± 20%—secondary; and 9% ± 8%—antral), follicular growth, and ovulation (as indicated by the presence of corpus luteum),²⁹ similar to that reported for cynomolgus monkeys (Macaca fascicularis).³⁰

Although vitrification of ovarian tissue cannot be considered as a routine procedure in reproductive medicine, some important advances have been attained in domestic (sheep, goats, cows, pigs, and dogs) and exotic (dasyurids and macaque monkeys) animals (Table 2). Despite these advances, cryopreservation of ovarian tissue is still a challenge, but its potential for biobanking genetic female material is incontestable.

In parallel, we know little about cryopreservation of isolated follicles, even in domestic animals, especially because of limitations related to the application of isolation methods that can damage early follicles, as discussed earlier.¹⁶ However, the procedure would have some advantages, including better cryoprotectant exposure. Regarding wild species, important results were reported for the rhesus monkey, but only for secondary follicles, which were isolated using an enzymatic method (DNase and collagenase), stored in a medium containing sucrose and ethylene glycol, and individually cryopreserved using a slow-freezing method. After thawing, these follicles were able to grow during in vitro culture, proving the efficiency of methods used for maintaining follicular viability.³¹ We therefore believe that cryopreservation of isolated follicles also stands as an alternative to be further explored for the creation of biobanks.

In Vitro Culture

In vitro culture of preantral follicles aims at mimicking the dynamics of the ovarian environment, cell communications, and interaction with secretory, hormonal, and growth factors.³² In recent years, attention has been devoted to the possibility of obtaining mature oocytes from the culture of frozen–thawed preantral follicles. Development of a system that allows in vitro growth of these follicles, resulting in oocytes capable of being matured and fertilized in vitro, would be of great importance. It would also contribute to long-term preservation of female germ cells and multiplication of endangered animals.²

Culture systems

Preantral follicles can be cultured in situ, enclosed into the entire ovary or into cortical fragments. This approach is most suitable for primordial and primary follicles because it ensures maintenance of the three-dimensional (3D) architecture of follicles and preserves interactions within follicular cells and between the follicle and surrounding stromal cells. However, when follicles develop to the antral stage, cortical tissues act as a barrier to medium perfusion into follicles.³² This system has been described for baboons (Papio sp.), in which primordial follicles within pieces of the ovarian cortex can survive and develop to secondary stages in serum-free culture.³³ This culture system also has already been successful for other species ranging from the Amur leopard (P. pardus orientalis)⁴ to peccary (P. tajacu).³⁴

Culture of isolated follicles is more adapted for more advanced follicular stages (secondary follicles at the minimum). While resting primordial and early growing primary follicles represent a larger follicle population, secondary follicles are less common and require more effort. In macaques, primordial or primary follicles can be isolated and maintain their viability when cultured in groups.¹⁶ Moreover, preliminary experiments conducted in rhesus macaques indicate that it is possible to grow individual primary follicles (80–120 μm in diameter) in vitro to small antral stages, which function in steroidogenesis, local factor production, and oocyte maturation.³⁵ This system allows better exposure to the medium and individual monitoring of follicles during the growth period. This is convenient to study substances involved in oocyte development, differentiation of granulosa cells, and regulation of autocrine/paracrine factors that control folliculogenesis.³⁶ However, this culture system requires a longer time period, a more sophisticated environment, and can be affected by isolation procedures (Table 3).

Table 3.

In Vitro Culture System, Duration, Media, and Results Achieved in Wild Animals

Species	In vitro culture system	Duration of in vitro culture	Medium used	Results achieved	Authors and year
Puma (P. concolor)	In situ	14 Days	Dulbecco's modified Eagle's medium	62.5% Viable follicles	Jewgenow and Stolte (1996)⁷
Amur leopard (Panthera pardus orientalis)	In situ	14 Days	Leibovitz	The overall number of follicles found in FCS cultured pieces (120 follicles)	Wiedemann et al. (2013)⁴
Rhesus monkeys	Encapsulated 3D culture system	≤30 Days	Alpha minimum essential	55% ± 15% follicles	Xu et al. (2009)¹²
Baboon	Encapsulated 3D culture system	14 Days	Alpha minimum essential	13 Oocytes exhibited germinal vesicle breakdown within 48 hours	Xu et al. (2011)¹⁵
Rhesus monkey	Encapsulated 3D culture system	40 Days	Alpha minimum essential	Healthy (n = 11) and mature (n = 1) oocytes	Xu et al. (2011)¹⁵
Rhesus monkey	Encapsulated 3D culture system	40 Days	Alpha minimum essential	Androgens promote preantral follicle development, but inhibit antral follicle growth and function	Rodrigues et al. (2015)⁴⁵
Rhesus monkey	Encapsulated 3D culture system	5 Weeks	Alpha minimum essential	Antrum formation	Baba et al. (2017)⁴⁷

3D, three-dimensional.

In a two-dimensional (2D) system, secondary follicles are cultured directly on a plastic surface or even on the surface of an extracellular matrix. In the gray short-tailed opossum (M. domestica), this culture system was able to support follicle growth; however, no development to the antral stage was observed.⁸ Similar results were reported for Tamar wallaby, in which the follicle diameter increased by 16% during culture, but there were no signs of antrum formation.⁹ Interestingly, it has been possible in marmosets (Callithrix jacchus) to develop mature oocytes (metaphase II [MII]) from secondary follicles (>85 μm) using a 2D system.³⁷

In the 3D system, follicles are embedded in an extracellular matrix, consequently follicles have no contact with the plate and do not adhere to it.^15,35 This is the best way to mimic natural follicular growth and regulations. This system also allows maintenance of the tridimensional follicular structure and prevents occurrence of degeneration during in vitro culture.³⁸ Currently, knowledge about development of preantral follicles in wild animals is more developed for nonhuman primates as biomedical models,³⁹ and substantial knowledge has been generated from studies with isolated secondary follicles cultivated in a 3D system. The encapsulated 3D culture system was first investigated in rhesus monkeys, starting from secondary follicles that then survived and grew in two hydrogel conditions (0.5% and 0.25% alginate) over 30 days of culture.¹² Then, an alginate application to nonhuman primates resulted in growth of small preantral follicles through the antral stage with production of ovarian steroids and local factors, as well as oocyte maturation,^12,15,35 being posteriorly adapted for capuchin monkeys (Sapajus apella).⁴⁰ To further improve 3D culture, secondary follicles from the baboon have been cultured in a fibrin–alginate matrix, grown to the antral stage, and their oocytes achieved meiotic maturation.¹⁵ Subsequently, the same results were obtained in the rhesus macaque (Macaca mulatta).^41,42 However, Xu et al.⁴¹ demonstrated that while preserving follicle survival, fibrin–alginate improves macaque primary, but not secondary, follicle development in terms of growth, steroidogenesis, anti-Müllerian hormone (AMH)/vascular endothelial growth factor (VEGF) production, and oocyte maturation.

Culture media

In vitro culture success of preantral follicles is tightly related to medium composition. In wild animals, studies comparing the efficiency of basic culture media are still scarce. Basic medium usually is a source of nutrients responsible for maintaining normal metabolic activities of different cell types. Those needs are species specific. For instance, in collared peccaries, it was recently demonstrated that TCM-199 is more efficient than α-MEM to promote preantral follicle development in vitro for 7 days.³⁴ Another study in the Amur leopard (P. pardus orientalis) reported better ovarian follicle development with 10% fetal bovine serum than with 0.1% bovine serum albumin during a 14-day culture period.⁴ Among the most common substances added to culture medium, FSH stands out due to its importance in folliculogenesis regulation. Positive effects on follicle survival and growth have been reported in short-tail gray opossums (M. domestica; 1.0–1.5 IU of FSH mL⁻¹),⁸ rhesus monkeys (M. mulatta; 500 mIU/mL of rhFSH),⁴³ and peccaries (P. tajacu; 50 ng/mL of FSH).³⁴ Interestingly, a negative effect on follicle integrity was shown in baboons (Papio sp.) when 10 or 100 mIU/mL of FSH was used.^15,35 Growth factors, such as bone morphogenetic protein 4 (BMP4), also are critical to promote in vitro preantral follicle development, as demonstrated in capuchin monkeys (S. apella). Moreover, the addition of eCG to culture media promoted positive effects on growth and survival of primate preantral follicles.⁴⁰

In the rhesus macaque, the presence of bovine fetuin in culture media associated with 5% of O₂ promotes a high preantral follicle survival rate, high percentage of healthy oocytes, and higher oocyte maturation rate.^15,35 Moreover, the association of FSH concentration and O₂ tension positively affects antral formation and production of VEGF-A in primate preantral follicles.⁴⁴

Addition of androgens (testosterone and dihydrotestosterone) to the culture medium alters follicle survival, growth, steroid and AMH production, and oocyte quality in vitro in a dose- and stage-dependent manner in nonhuman primates. However, excessive levels of androgens may have negative impacts on primate folliculogenesis.⁴⁵ According to Ting et al.,⁴⁶ androgens appear to be a survival factor, but hinder antral follicle differentiation, while estrogen appears to be a factor in survival and growth at the preantral and early antral stages. Progesterone may not be essential during early folliculogenesis in primates.⁴⁶ However, their growth-promoting effects are limited to preantral and early antral stages.⁴⁷ These authors reported that the primate follicle pool is heterogeneous and differs between animals, therefore even though only secondary follicles were selected, follicle growth and developmental outcomes might differ from one animal to another.

Addition of 25 pg/mL of 1,25-dihydroxy vitamin D3 (VD3) increases preantral follicle survival and maintains anti-Mullerian production by antral follicles, while 100 pg/mL of VD3 improves antral follicle growth. VD3 supplement promoted oocyte growth in in vitro developed follicles in primates, but dosage has to be adapted to the follicular stage.⁴²

It is critical to continue research to better understand which factors are involved in the initial phase of folliculogenesis to define a culture system that provides oocytes suitable for application in other biotechnology applications.

Xenografting

Ovarian tissue transplantation has been explored as a promising alternative.⁴⁸ Results of xenotransplantation to immunosuppressed rodents span from simple activation of primordial follicles to growth until the early antral stage in several species (marmosets,^21,49 elephants,⁵⁰ wallabies,⁵¹ wombats,⁵² lionesses,⁵³ and baboons²⁹). However, maturation of oocytes obtained from grafted ovarian tissues has not been reported yet. While fertility preservation using ovarian tissue banks and transplantation is still in the experimental phase, it is increasingly investigated in several groups of wild mammals such as felines⁵³ and nonhuman primates.²⁹ In wild rodents such as agoutis, viability of ovarian tissues after vitrification and xenografting in SCID mice was recently demonstrated with resumption of ovarian activity 40 days after the transplant.³ The main challenge still is to establish the most appropriate technique to be used in each species (allotransplantation or xenotransplantation), the size of the graft, site of transplantation, and eventual maturation of resulting oocytes.²

Final Considerations

In addition to the lack of precise knowledge about reproductive physiology in many wild species, there are still few comparative studies on follicular development. Initial studies related to the recovery of primordial and primary follicles have been conducted on only few wild species, but results are promising to improve culture systems and promote development of these follicles. Regarding cryopreservation, protocols developed to date are efficient for storing many preantral follicles in some wild animal species; however, it is necessary to standardize protocols for a more optimal storage in germplasm banks. These systematic approaches will greatly contribute to preservation of rare species.

Footnotes

Acknowledgments

This study was financed, in part, by the CAPES (Financial Code 001). A.R.S. is a recipient of a grant by CNPq.

Author Disclosure Statement

No competing financial interests exist.

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